The crucial role of cyclic AMP in cell proliferation and differentiation has been well-described. The addition of retinoic acid (RA), cholera toxin (CT), or 8-Br cyclic AMP (8-BrcAMP) to a human promyelocytic leukemia cell line (HL60) in culture results in a marked differentiation of the cells to a mature myeloid phenotype, while the addition of the phorbol diester 12-0-tetradecanoyl phorbol-13-acetate (TPA) results in the differentiation of the cells into macrophages. We have detected cAMP-dependent and -independent altered phosphorylations of specific endogenous proteins during RA-\and TPA-induced differentiation of HL60 cells which appear to be specific for each pathway of differentiation. We have noted marked changes in the cAMP-dependent and -independent protein kinase activities which are different in cells differentiated along the macrophage pathway from those differentiated to the mature myeloid phenotype. Retinoic acid and agents raising intracellular cyclic AMP synergistically induced myeloid differentiation. This project is concerned with further investigating the specificity and role of the endogenous phosphoprotein patterns and cAMP-dependent and -independent protein kinases in HL60-induced differentiation by: (1)\investigation of the nature of the synergism between RA and cyclic AMP and the possibility of synergism between cAMP and other classes of differentiating agents; (2)\study of the phosphoprotein patterns obtained both in vivo and in vitro in various subcellular fractions of HL60 cells differentiated by means of RA, CT, 8-BrcAMP, and TPA to determine (a)\whether RA alters the phosphorylation of the same proteins as CT, 8 BrcAMP, (b) if synergistic induction synergistically alters protein phosphorylation, (c) if altered phosphorylation is an early event specifically related to the differentiation of HL60 cells along specific pathways; (3)\study of phosphoprotein patterns in patient-derived myelogenous leukemia cells differentiated by RA and 8-BrcAMP to compare them to the HL60 cell line; and (4)\investigation of cyclic AMP-dependent and protamine kinases in HL60 cells induced to differentiate and the correlation of altered activity, distribution, etc., to altered phosphoprotein patterns. It is hoped that understanding of this biochemical process of differentiation will allow us to improve current clinical treatments. (M)